1. Field of the Invention
The present invention relates generally to pharmaceutical compositions and medicaments comprising dimethyl sulfoxide (DMSO) and/or related compounds in combination with one or more other compounds, such as L-arginine, fructose 1,6-diphosphate, L-lysine, L-aspartate, and urea.
2. Description of the Related Art
Traumatic brain injury and stroke generally cause a reduction in cerebral blood flow (CBF), which may cause additional damage to the brain. Applicant believes that there are presently no known therapeutic agents which increase CBF in a sustained fashion (for at least several days) after traumatic brain injury. (Narayan K, and NIH Collaborative Committee. Clinical trials in head injury. J. Neurotrauma. 2002; 19(5):503-57, herein incorporated by reference).
Nitric oxide (NO) is a multifunctional messenger molecule that has a prominent role in the regulation of CBF and cell-to-cell communication in the brain. Its highest levels in the body is found in neurons. NO is synthesized from L-arginine by a family of enzymes called NO synthases (NOS). Release of NO from cerebral endothelial cells to produce vasodilation is a fairly well established reaction. NO has been shown to diffuse towards the lumen of blood vessels in humans where it helps maintain blood fluidity, and by inference, reduce blood viscosity, thus improving blood flow. (Moncada, S., Palmer, R. M., and Higgs, E. A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev 1991; 43, 109-142; Ignarro L, Napoli C. Novel features of nitric oxide, endothelial nitric oxide synthase, and atherosclerosis. Curr Atheroscler Rep. 2004 July; 6(4):281-7, herein incorporated by reference).
Arginine is a basic amino acid that plays several pivotal roles in cellular physiology. Like any amino acid, it is involved with protein synthesis, but it is also intimately involved with cell signaling through the production of NO and cell proliferation through its metabolism to ornithine and the other polyamines. Because of these multiple functions, arginine is an essential substrate for healing processes involving tissue trauma. Numerous studies have shown that arginine supplementation can lead to normalization or improvement of wound healing. (Barbul A. Arginine: biochemistry, physiology, and therapeutic implications. J Parent Enteral Nutr 1986; 10:227-238; Cheman L. L-Arginine and Free Radical Scavengers Increase Cerebral Blood Flow and Brain Tissue Nitric Oxide Concentrations after Controlled Cortical Impact Injury in Rats. Journal of Neurotrauma, January 2003, 20 (1): 77-85; Hlatky R. The Role of Endothelial Nitric Oxide Synthase in the Cerebral Hemodynamics after Controlled Cortical Impact Injury in Mice. Journal of Neurotrauma, 2003, 20 (10): 995-1006, all herein incorporated by reference).
Studies have shown that L-arginine administration after experimental traumatic injury in mice increased CBF post-injury. L-Arginine administration also resulted in a reduction in contusion volume in the L-arginine treated mice. The likely explanation for these results is that the increase in CBF was beneficial to the outcome of the head injury in these animals, and such action is mediated by vascular NO. These findings suggest an important role for vascular NO produced by endothelial NO synthase (eNOS) in the preservation of cerebral blood flow in contused brain following traumatic injury, and in the improvement in cerebral blood flow with L-arginine administration. Normal synthesis of vascular NO from L-arginine is achieved by the action of eNOS and specific co-factors nicotinamide adenine dinucleotide phosphate (NADPH) and tetrahydrobiopterin (BH4) in the endothelium.
L-arginine is a non-toxic, inexpensive, natural amino acid that can be given in high doses orally for prolonged periods of several months or intravenously for several weeks. (Piatti P, Fragasso G, Monti L D, Setola E, Lucotti P, Fermo I, Paroni R, Galluccio E, Pozza G, Chierchia S, Margonato A. Acute intravenous L-arginine infusion decreases endothelin-1 levels and improves endothelial function in patients with angina pectoris and normal coronary arteriograms: correlation with asymmetric dimethylarginine levels. Circulation. 2003; 107(3):429-36, herein incorporated by reference).
Arginine is a dibasic amino acid, and is found in many proteins in the body. Its metabolism is intimately tied to several metabolic pathways involved in the synthesis of urea, NO, polyamines, agmatine, and creatine phosphate. (FIG. 1). Arginine can be provided via nutritional intake, via new synthesis, or via systemic administration, for example, intravenously. About 50% of the ingested arginine is released into the portal circulation. The other part is directly utilized in the small bowel. The physiological uptake of arginine and citrulline by the liver is low because the liver does not express large amounts of the cationic transporter for the basic amino acid arginine. Therefore, most of the portal venous arginine and citrulline enters the systemic circulation and serves as substrate for extrahepatic tissues. The kidney metabolizes citrulline into arginine (the “intestinal-renal axis”) and exports arginine into the systemic circulation. (FIG. 1).
The average nutritional arginine uptake is approximately 5-6 g/day. Standard rodent laboratory chow diets contain about 1% L-arginine, which corresponds to an average intake of 1 g arginine/kg body weight/day. Arginine-deficient rats subjected to minor trauma lose significantly more weight and are more likely to experience mortality when compared to arginine-repleted animals.
Arginine catabolism occurs via several enzymatic pathways (FIG. 1). The two major catabolic pathways during healing after trauma are degradation via NO synthase (NOS) isoforms and via the two arginase isoforms. Both pathways deplete extracellular arginine concentrations in the wound milieu, thus rendering arginine an essential amino acid for wound healing. The current interest in L-arginine is due mainly to its close relation with the important signal molecule NO.
The major isoform of NOS activation during healing after trauma is inducible nitric oxide synthase (iNOS), which generates larger amounts of NO than the constitutive isoforms (endothelial NOS and neuronal NOS). Major sources of iNOS are macrophages but also fibroblasts, endothelial cells, and keratinocytes. Strong counter-regulating mechanisms exist between the two catabolic pathways. Intermediates and end products of each pathway can reciprocally inhibit each other. Each pathway is stimulated by a well-defined set of cytokines that in turn also down-regulates the alternate pathway.
Arginase exists in two different isoforms. Arginase I is the cytosolic “hepatic” isoform that is also present in wound-derived fibroblasts. Arginase II, the mitochondrial extrahepatic isoform, is present in many other cell types such as macrophages, kidney, breast tissue, and enterocytes. The two isoforms are encoded by different genes and have their own distinct regulation. It is unclear which isoform, if any, plays the predominant role in the wound environment.
The main source of vascular NO in mammals is derived from eNOS contained within the endothelial cells. The loss or uncoupling of eNOS impairs cerebrovascular function in part by promoting vasoconstriction, platelet aggregation, smooth muscle cell proliferation, leukocyte adhesion and greater endothelial-immune cell interaction. Vascular NO production from the endothelium is regulated by eNOS enzyme activity and/or NOS gene expression. (Kubes P. and Granger, D. N. (1992). Nitric oxide modulates microvascular permeability. Am. J. Physiol. 262, H611-H615, herein incorporated by reference).
Besides the key role vascular NO plays in vascular tone, blood pressure and vascular homeostasis, it also acts to inhibit platelet and leukocyte adhesion to the endothelium, a process that may down-regulate pro-inflammatory events. (Kubes P., Kanwar S., Niu X. F. (1993). Nitric oxide synthesis inhibition induces leukocyte adhesion via superoxide and mast cells. FASEB J. 7, 1293-1299, herein incorporated by reference).
When trauma to the brain reduces cerebral blood flow (CBF), formation of reactive oxygen species (ROS) at the injury site may induce a deficiency in tetrahydrobiopterin (BH4), a rate limiting step in eNOS synthesis, resulting in eNOS uncoupling and reduced release of vascular NO. Reduced vascular NO is reported to involve many changes including: endothelial cell (EC) shape changes, mitochondrial stress, reduced eNOS, impaired glucose transporter 1 (thus lowering glucose delivery to brain cells), tumor necrosis factor-alpha (TNF-alpha) activation, neutral factor-kappa B (NF-kB) translocation from cytosol to nucleus and activation of transcription inflammatory genes, release of the powerful vasoconstrictor endothelin-1 (ET-1), migration of vascular smooth muscle cells (VSMC) leading to the formation of vessel wall plaques, activation of hypoxic inducible factor-1alpha (HIF-1alpha), increase of vascular adhesion molecules (VCAM), increased beta peptide angiopathy, excess free radical formation including hydrogen peroxide (H2O2) and superoxide anion (SO−), impairment of the angiogenic vascular endothelial growth factor (VEGF) and persistent shear-stress on vessel walls. (de la Torre J C, Stefano G B. Evidence that Alzheimer's disease is a microvascular disorder: Role of constitutive nitric oxide. Brain Res Rev. 34:119-136, 2000, herein incorporated by reference).
Vascular NO therefore, acts as an antiatherogenic, antithrombotic and anti-ischemic molecule. No does this by reducing oxidative stress, by preventing platelet aggregation and by stimulating angiogenesis via vascular endothelial growth factor (VEGF) while reducing shear stress on the vessel wall.
The increased synthesis of vascular NO by L-arginine appears to be a logical approach for the treatment of severe traumatic brain injury, acute ischemic stroke, and neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, subacute sclerosing panencephalitis, vascular dementia, multiple sclerosis, assorted neuropathies, Huntington's disease, amyotrophic lateral sclerosis (ALS) and leukodystrophies.
L-arginine produces peak plasma levels approximately 1-2 hours after oral administration. The most common adverse reactions of higher doses, from 15 to 30 grams daily, are nausea, abdominal cramps and diarrhea. (Visser J J, Hoekman K. Arginine supplementation in the prevention and treatment of osteoporosis. Med. Hypotheses. 1994 November; 43(5):339-42, herein incorporated by reference).
Additionally, L-arginine given as a continuous intravenous infusion for 120 minutes at a rate of 0.125 g/min, for angina pectoris, was able to reduce the levels of endothelin-1, one of the most powerful vasoconstrictors known and also lowered the serum levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of eNOS.
DMSO has been shown to increase CBF in a variety of brain injuries including stroke and head trauma in animals and humans. The combination of DMSO with fructose 1,6-diphosphate has been reported to of benefit to victims of acute and chronic human stroke. The mechanism of DMSO action for increasing CBF after brain injury is not clear but may be due to its ability to: i) reduce cerebrovascular reactivity, ii) deaggregate platelets in blood vessels thus augmenting blood fluidity by decreasing blood viscosity and iii) reducing intracranial pressure, thus allowing compressed blood vessels in brain tissue to return to a more normal hemodynamic state. DMSO is not known to affect vascular nitric oxide, ADMA or endothelin-1. (de la Torre, J. C. and Surgeon, J. W.: Dexamethasone and DMSO in cerebral infarction. Stroke, 7:577-583, 1976; de la Torre, J. C., Kawanaga, H. M., Goode, D. J., Johnson, C. M., Kajihara, K., Rowed, D. W. Mullan, S.: Dimethyl sulfoxide in CNS trauma. Ann. N.Y. Acad. Sci., 243:362-389, 1975; Brown F D, Johns L M, Mullan S. Dimethyl sulfoxide in experimental brain injury, with comparison to mannitol. J. Neurosurg. 1980 July; 53(1):58-62; Karaca M, Kilic E, Yazici B, Demir S, de la Tone J C. Ischemic stroke in elderly patients treated with a free radical scavenger-glycolytic intermediate compound. Neurol Res, 24:73-80, 2002; Karaca, M., Bilgin, U., Akar, M. and de la Torre, J. C.: Dimethyl sulfoxide lowers ICP after closed head trauma. Eur. J. Clin. Pharmacol., 40:113-114, 1991, all herein incorporated by reference).
Ischemia has been proposed to cause an excess increase in the extracellular concentration of glutamate, an excitotoxic amino acid, in the central nervous system. (Benveniste H, Drejer J, Schousboe A, Diemer N H: Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem 1984; 43: 1369-74, herein incorporated by reference).
The increased glutamate in turn triggers a surplus influx of calcium ion (Ca2+) from the extracellular space into the cytosol, resulting in the initiation of a neuronal cell death cascade. The extracellular glutamate concentration is tightly regulated by release from presynaptic membranes and uptake by postsynaptic membranes and glia. This regulation is closely linked to alterations in intracellular free calcium concentration; namely, an increase in intracellular Ca2+ may enhance glutamate release from glutamatergic neurons and astrocytes. Therefore, controlling the extracellular glutamate and intracellular Ca2+ concentrations could be a promising strategy for alleviating ischemic and traumatic neuronal damage. (Kristian T, Siesjö; B K: Calcium in ischemic cell death. Stroke 1998; 29: 705-18; Rossi D J, Oshima T, Attwell D: Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature 2000; 403: 316-21; Bezzi P, Carmignoto G, Pasti L, Vesce S, Rossi D, Rizzini B L, Pozzan T, Volterra A: Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 1998; 391: 281-292, all herein incorporated by reference).
It has been reported that concentrations of DMSO to which neurons are typically exposed in experimental studies and in human patients (0.5-1.5%) inhibit glutamate responses in hippocampal neurons. DMSO suppresses, in a rapidly reversible manner, electrophysiological responses and calcium influx induced by glutamate, NMDA (N-methyl-1-aspartate), and AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate). Moreover, DMSO can prevent excitotoxic death of the neurons induced by glutamate. The findings have important implications for the use of DMSO as a therapeutic agent that involve glutamatergic excitotoxicity after head trauma. These findings by an NIH group of investigators identify a mechanism that might explain the beneficial clinical effects of DMSO on CNS neurons and suggest a potential use for DMSO in the treatment of excitotoxic traumatic and neurodegenerative conditions. (Lu, C., and M. P. Mattson. 2001 July. Dimethyl sulfoxide suppresses NMDA- and AMPA-induced ion currents and calcium influx and protects against excitotoxic death in hippocampal neurons. Exp Neurol 170:180-185; Marshall L F, Camp P, Bowers S. Dimethyl sulfoxide for the treatment of intracranial hypertension. J Neurosurg 1984; 14: 659-663, herein incorporated by reference).